Recombinant human tumor necrosis factor (rH-TNF) is a cytokine with direct antitumor properties. In a phase I trial we continuously infused rH-TNF for 24 hours. We gave a total of 115 courses of therapy to 50 patients. Doses ranged from 4.5 to 645 micrograms of rH-TNF/m2. Systemic toxicity, including fever, chills, fatigue, and hypotension, increased with the dose of rH-TNF administered. Doses greater than 454 micrograms/m2 frequently caused severe lethargy and fatigue, which precluded hospital discharge of the patient at the completion of therapy. The dose-limiting toxicity was hypotension, and five patients treated at the two highest dose levels required dopamine treatment. Other organ-specific toxicity was modest and spontaneously resolved after 48 hours. The 24-hour infusions of rH-TNF were associated with significant decreases in serum cholesterol and high-density lipoprotein levels. Pharmacokinetic studies using an enzyme-linked immunosorbent assay demonstrated peak plasma rH-TNF levels of 90-900 pg/mL. Despite continuous infusion of rH-TNF, no steady-state level was achieved. The recommended phase II dose for rH-TNF as a 24-hour continuous infusion is 545 micrograms/m2.
Regulation of tumor necrosis factor (TNF) gene expression was investigated in resting human monocytes and in 12-0-tetradecanoylphorbol-13-acetate (TPA) activated monocytes. TNF transcripts were undetectable in resting monocytes. However, in TPA-activated monocytes, TNF mRNA was first detectable by 3 h and reached maximal levels by 12 h of drug exposure. Using run-on transcription assays, the TNF gene was transcriptionally inactive in resting monocytes, but was rapidly activated after TPA exposure. The protein synthesis inhibitor, cycloheximide (CHX), had no detectable effect on levels of TNF transcripts in resting monocytes, while this agent superinduced the level of TNF mRNA by 50-fold in TPA-activated cells. TPA activated monocytes were also exposed to actinomycin D and/or CHX to determine whether transcriptional or posttranscriptional control of TNF gene expression was responsible for the induction of TNF transcripts. After 1 h of actinomycin D treatment, the amount of TNF transcripts was reduced by 75%. In contrast, no difference in TNF mRNA levels was observed in TPA-activated monocytes exposed to CHX alone or CHX in combination with actinomycin D. These findings indicated that CHX prevented the degradation of TNF mRNA by inhibiting the synthesis of a labile protein. Run-on transcription assays performed on cells exposed to either TPA or the combination of TPA and CHX further indicated that CHX treatment increased transcription of the TNF gene. Thus, TNF gene expression is controlled at the transcriptional level in resting human monocytes, while both transcriptional and posttranscriptional events regulate the level of TNF transcripts in TPA-activated cells.
Recombinant human tumor necrosis factor (rH-TNF) is a cytotoxic monokine with pleiotropic effects. A phase I trial of rH-TNF was initiated using a five-day continuous intravenous (IV) infusion repeated every 28 days. Thirty-eight courses of therapy were administered to 19 patients. The starting dose was 5 X 10(4) U/m2/d, with escalations to 1.0 X 10(5), 2.0 X 10(5), 2.4 X 10(5), and 3.0 X 10(5) U/m2/d. Systemic side effects, including fever, chills, hypotension, fatigue, anorexia, and headaches, were mild and self-limiting. At the maximum tolerated dose of 3.0 X 10(5) U/m2/d, dose-limiting hematologic toxicity was manifested by transient thrombocytopenia and leukopenia. Elevated bilirubin levels were also seen at the higher dose levels. Lipoprotein analysis demonstrated that the five-day treatment with rH-TNF was associated with decreases in high-density lipoproteins, as well as increases in triglycerides and very-low-density lipoproteins. Pharmacokinetic studies using an enzyme-linked immunosorbent assay (ELISA) test indicated plasma rH-TNF levels less than 0.2 U/mL. The recommended phase II dose of rH-TNF administered as a five-day continuous infusion is 2.4 X 10(5) U/m2/d.
Colony‐stimulating factor 1 (CSF‐1) is required for the survival, proliferation and differentiation of monocytes. We previously demonstrated that the CSF‐1 receptor is linked to a pertussis toxin‐sensitive G protein and that the induction of Na+ influx by CSF‐1 is a pertussis toxin‐sensitive event. The present studies have examined activation of protein kinase C as a potential intracellular signaling event induced by the activated CSF‐1 receptor. The results demonstrate that CSF‐1 stimulates translocation of protein kinase C activity from the cytosol to membrane fractions. This activation of protein kinase C was sensitive to pretreatment of the monocytes with pertussis toxin. Lipid distribution studies demonstrated that phosphatidylcholine (PC) is the major phospholipid in human monocytes. Moreover, the results indicate that CSF‐1 stimulation is associated with decreases in PC, but not in phosphatidylinositol (PI), levels. The absence of an effect of CSF‐1 on PI turnover was confirmed by the lack of changes in inositol phosphate production. In contrast, CSF‐1 stimulation was associated with increased hydrolysis of PC to phosphorylcholine and diacylglycerol (DAG) in both intact monocytes and cell‐free assays. Furthermore, the increase in PC turnover induced by CSF‐1 was sensitive to pertussis toxin. The results also demonstrate that the induction of Na+ influx by CSF‐1 is inhibited by the protein kinase C inhibitors staurosporine and the isoquinoline derivative H7, but not by HA1004.(ABSTRACT TRUNCATED AT 250 WORDS)
The treatment of human HL-60 promyelocytic leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with induction of tumor necrosis factor (TNF) transcripts. The study reported here has examined TPA-induced signaling mechanisms responsible for the regulation of TNF gene expression in these cells. Run-on assays demonstrated that TPA increases TNF mRNA levels by transcriptional activation of this gene. The induction of TNF transcripts by TPA was inhibited by the isoquinolinesulfonamide derivative H7 but not by HA1004, suggesting that this effect of TPA is mediated by activation of protein kinase C. TPA treatment also resulted in increased arachidonic acid release. Moreover, inhibitors of phospholipase A2 blocked both the increase in arachidonic acid release and the induction of TNF transcripts. These findings suggest that TPA induces TNF gene expression through the formation of arachidonic acid metabolites. Although indomethacin had no detectable effect on this induction of TNF transcripts, ketoconazole, an inhibitor of 5-lipoxygenase, blocked TPA-induced increases in TNF mRNA levels. Moreover, TNF mRNA levels were increased by the 5-lipoxygenase metabolite leukotriene B4. In contrast, the cyclooxygenase metabolite prostaglandin E2 inhibited the induction of TNF transcripts by TPA. Taken together, these results suggest that TPA induces TNF gene expression through the arachidonic acid cascade and that the level of TNF transcripts is regulated by metabolites of the pathway, leukotriene B4 and prostaglandin E2.
Tumor necrosis factor (TNF) is a polypeptide cytokine that is cytotoxic to some but not all tumor cells. The basis for resistance to the cytotoxic effects of this agent remains unclear. We have studied the development of TNF resistance in human ZR-75-1 breast carcinoma cells. ZR-75-1 cells have undetectable levels of TNF RNA and protein. However, TNF transcripts are transiently induced in these cells by exposure to recombinant human TNF. This induction of TNF RNA is associated with production of TNF-like protein in cell lysates and culture supernatants. Stable resistance to TNF-induced cytotoxicity develops when ZR-75-1 cells are exposed to increased concentrations of TNF. The TNF-resistant cells, designated ZR-75-1R, continuously express TNF transcripts and a TNF-like protein. Furthermore, ZR-75-1R cell supernatants contain cytotoxic activity that is abrogated by polyclonal antibody against TNF. The ZR-75-1R cells also possess TNF receptors that are occupied or down-regulated by the TNF-like protein. These findings thus suggest that (') TNF induces TNF transcripts and production of a TNF-like protein in ZR-75-1 cells and (it) resistance to TNF-induced cytotoxicity is associated with stable TNF expression.Tumor necrosis factor (TNF) is a polypeptide cytokine that exerts a wide variety of biological effects (1). TNF was originally identified by its ability to cause hemorrhagic necrosis in subcutaneous murine tumors (2). Subsequent studies showed that TNF is cytotoxic to certain murine and human tumors, both in vitro and in vivo (1-3).The cytotoxic effects of TNF appear to be cell cycledependent. TNF causes accumulation ofcells in G2 phase and cytolysis in late stages of mitosis (4). This cytolytic effect is enhanced by inhibitors of RNA and protein synthesis (5, 6). Tumor cells sensitive and resistant to TNF-induced cytotoxicity have similar numbers of cell surface receptors. Furthermore, both sensitive and resistant cells internalize and degrade TNF after receptor binding (7). Thus, cell surface receptors appear to be necessary but not sufficient for TNF cytotoxicity (8)(9)(10). The basis for the sensitivity or resistance of transformed cells to TNF remains unknown.Exposure of TNF-sensitive L-929 mouse fibroblasts to TNF results in the development of stable TNF resistance (11). Paradoxically, this TNF-resistant subline produces a TNF-like protein that is cytotoxic to the parent L929 cell line. We recently demonstrated that certain human epithelial tumor cell lines inherently resistant to TNF also produce both TNF mRNA and protein (unpublished work). These findings suggested that production of TNF may be associated with TNF resistance. The present studies investigated this relationship in human breast carcinoma cells sensitive to the cytotoxic effects of TNF in vitro. The results show that TNF treatment induces both TNF expression and resistance to TNF cytotoxicity. METHODSCell Culture. The ZR-75-1 human breast carcinoma cells were obtained from the American Type Culture Collection and grown in RPMI 1640 ...
Tumor necrosis factor (TNF) is a monokine with in vitro cytotoxicity for some but not all tumor cells. The basis for sensitivity and resistance to the antitumor effects of this agent remains unclear. The present studies have monitored the effects ofTNF on 14 epithelial tumor cell lines. Eleven of these cell lines were resistant to the growth inhibitory effects of TNF (50% inhibitory concentration > 1,000 U/ml). 12 of the 14 tumor cell lines has detectable levels of high affinity cell surface TNF binding sites, thus suggesting that resistance was not often due to the absence of cell surface TNF receptors. Northern blot analysis demonstrated that three of the eleven resistant cell lines expressed detectable levels of TNF mRNA. Furthermore, both sensitive and resistant epithelial tumor cells had the capacity to express TNF transcripts in the presence of the protein synthesis inhibitor, cycloheximide. Finally, the presence of TNF expression at the RNA level is shown to be associated with the production of a TNF-like protein in the resistant Ov-D ovarian carcinoma cells. These findings suggest that certain human epithelial tumor cell lines inherently resistant to TNF also express this cytokine.
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